The present invention relates primarily to a test for diagnosing the existence of certain disease states or physiological conditions in mammals based on the collection and analysis of apocrine sweat.
It is known that certain disease states or variations from normal physiological conditions can be detected or monitored by analyzing body fluids for variations in constituents of those body fluids. Blood is a highly complex suspension with many elements, which can be measured and analyzed to measure normal homeostasis and variations from normality. Blood is frequently analyzed for variations of various constituents (cholesterol, glucose, etc.), as well as its cellular constituents, as indicators of possible abnormal health conditions. In a like manner, a urine analysis may be used to detect various chemical imbalances, pregnancy or the presence of various legal or illegal drugs by testing for metabolites of the target drug. In addition, there are numerous examples of calorimetric test strips, typically used on urine, for indicating various concentrations of normal fluid constituents or environmental contaminants such as various metals, glucose, protein and ketones. The strips provide a visible indication of a threshold level of normally present reference analyte such as IgG, albumin or the like.
Diagnostic studies of chemical imbalances in other body fluids, such as sweat, can also be indicative of different disease states. For example, an increase in the level of chloride ion in sweat is indicative of cystic fibrosis. U.S. Pat. No. 3,552,929 describes a band-aid type test patch for collecting perspiration. The test patch is applied to an accessible skin surface for collecting eccrine, not apocrine sweat. It is then removed and subjected to a series of analytical tests to determine the concentration of chloride ions as an indicator of cystic fibrosis. U.S. Pat. No. 4,329,999 to Phillips is a further example of a sweat collection device for the detection of drug or alcohol use. The patch was applied to shaved skin surfaces (ankle or calf) which do not have apocrine sweat glands and remained in place for ten days. U.S. Pat. No. 4,706,676 discloses a third example of a dermal patch for detecting exposure to various different environmental chemicals. No attempt was made to collect apocrine sweat.
U.S. Pat. No. 5,676,144 to Schoendorfer and related patents (U.S. Pat. Nos. 5,638,815; 5,465,713; 5,438,984; 5,441,048; 5,203,327 and 4,957,108) describe skin patches which contain various immobilized specific binding agents for certain reference analytes as well as compounds to be detected. Schoendorfer recognizes that there are both eccrine and apocrine sweat glands, that perspiration can be either sensible or insensible and that various locations on the body sweat at different rates. However, he fails to recognize the unique diagnostic function of apocrine sweat glands and proceeds to collect eccrine sweat from the chest because large quantities of sweat can be collected. After a sufficient volume of sweat is collected the patches are then removed and subjected to various different laboratory procedures to detect the presence or increased levels of various substances such as creatine kinase MB (CKxe2x80x94MB). CKxe2x80x94MB is released from cardiac muscle during myocardial infarction and other cardiac stress and therefore is an indicator of cardiac injury. As indicated in U.S. Pat. No. 5,203,327, many chemical species detectable in blood or urine, such as sodium, calcium, chloride and potassium, are also detectable in lower concentrations in sweat. Table 1 of the ""327 patent also lists numerous other normally occurring components detectable in various concentrations in sweat. Affinity chemistry can be developed and incorporated in skin patches for monitoring the concentration of these various chemicals. In addition, various metabolites of drugs of abuse such as THC, morphine, cocaine or methadone can be detected in sweat. However, with the exception of colorimetric indication of a normally present reference compound, such as IgG or albumin, which is used to provide an indication that a minimum volume of sweat has entered the patch, the compounds of interest are merely collected by the patch (U.S. Pat. No. 5,203,327, col. 16, lines 4-30). Detection and analysis of the target component is then performed in a laboratory setting.
The FDA has approved sweat collection patches for use in drug abuse screening programs to test for amphetamines, cocaine, marijuana, PCP, heroin meth-amphetamines, their metabolites and other opiates. Manufacturers of these products include the Sudormed Sweat Specimen Container, made by Sudorned, of Santa Anna, Calif., the EIA Micro-Plate Assay, made by SolarCare Technologies Corporation, Bethlehem, Pa. and the PharmaChek(trademark) Sweat Patch from Pharmachem Laboratories Inc. of Menlo Park, Calif. These patches are waterproof, adhesive pads about the size of a playing card which is worn on the back, upper arm or lower chest. As such, it collects eccrine sweat. The patch has a tamper-proof feature so it can be applied only once and not removed and reapplied later. It is not designed for use by individuals; instead it is intended for use by trained drug abuse testing professionals in clinical and rehabilitation centers. After removal, the test patch is sent to a test laboratory where the presence and quantity of the target compound is detected by a special laboratory assay. It must be worn for 5 to 14 days to collect an adequate quantity of chemicals indicative of drugs of abuse used during that period. Sweat testing for drugs of abuse provides a convenient and considerably less invasive method for monitoring drug exposure than blood or urine. Typically, ELISA immunoassay is used to detect opiates collected on the patch from eccrine sweat and GC/MS is used to determine specific opiates. For comparison urine specimens are subjected to qualitative analysis by EMIT. However, the percentage of false negative results with the patches indicates that weekly sweat testing may be less sensitive than thrice weekly urine testing in detecting opiate use. (Huestis M. A., Cone, E. J., Wong, C. J., Umbricht, A., and Preston, K. L. Journal of Analytical Toxicology, 24, pp. 509-521, 2000). This is most likely because those skilled in the art selected an inappropriate source of sweat (eccrine sweat), not recognizing the unique diagnostic capacity of apocrine sweat, Further, because of the extended wear requirement (up to 14 days), the ability to rapidly test for the presence of drugs using presently available sweat collection patches and procedures is not possible.
Besides the use of skin patches to collect sweat, there are also several skin patch designs for use to deliver chemicals to the skin surface, such as anti-infectives or allergens, for treating various maladies or for analyzing sensitivity to various allergens.
It is also known to collect an analyte on a test strip and then perform various tests on the analyte in a laboratory environment. U.S. Pat. No. 5,459,080 sets forth numerous prior techniques for analysis, both quantitatively and qualitatively, of the presence of various compounds found in body fluids. In particular, the use of specific binding reagents is discussed. Such analytes include, but are not limited to, antibodies, antigens, drugs and hormones. One technique for analyzing these materials, referred to as immunoassay procedures, uses antibodies and antigens, i.e. immunoreactants, which are capable of binding with each other, creating a highly specific reaction product indicative of the presence of the antigen. To use these techniques the analyte must first be collected and then transported to a laboratory where the analytical procedure can be performed. Typically, the analyte on the test strip is then contacted with an indicator reagent which is then subjected to a detection means. The requirement for such a procedure results in a major deficiency for diagnostic test strips as it is often desirable to be able to read the test strip on the patient or immediately upon removal of the test strip without performing a further analytical procedure.
Other patents directed to test strip detection systems include U.S. Pat. No. 5,900,379 to Noda et al, U.S. Pat. No. 5,965,458 to Kouvonen et al and U.S. Pat. No. 5,998,221 to Milleck et al.
In a related approach, to test for pregnancy, monoclonal antibodies are used to bind to specific chemicals, such as human chorionic gonadotropin. A detector agent is then added to the urine/antibody combination to produce a visible color change.
It is also known that certain cancers generate marker compounds, or levels of such compounds elevated above normal, which can be detected in blood and quantified. Elevated levels of prostate specific antigen (PSA) may be an indication of the presence of prostate cancer. Likewise elevated levels of prostate acid phosphatase are found in some patients with prostate cancer. The protein NPM66 is a marker for breast cancer. CA 125 is produced by a variety of cells; elevated levels are found in many women with ovarian cancer. While human chorionic gonotropin (HCG) is normally produced by the placenta during pregnancy, it can also indicate a rare form of uterine cancer. Porter-Jordan et al (Hematol. Oncol. Clin. North Amer. 8, May, 1993, 28-44) suggests that tumerigenesis and tumor growth can be assessed by measuring cell proliferation markers (Ki67, cyclin D1 and proliferating epidermal nuclear antigen (PCNA)). Elevated levels of urokinase plasminogen activator (upa) has been reported in patients with breast carcinomas. CA 15-3 is shed by breast tumor cells into the blood stream. While the clinical significance is not clear, the FDA has approved test kits to measure CA 15-3-like antigen for the early detection of recurrent disease in breast cancer patients. CA 27-29 is also frequently present in breast cancer patients, as well as in the blood of patients with colon, stomach, kidney, lung, ovary, pancreas, uterus or liver cancer. CA 19-9 has been identified in patients with colorectal, pancreatic, stomach and bile duct cancer. Measurement of neuron-specific enolase (NSE) has been tracked as a marker for neuroblastoma, small cell lung cancer, Wilms"" tumor, and cancers of the thyroid, kidney, testicle and pancreas. Her2/neu (also known as v-erb2) is amplified in 20-30% of breast cancer patients. It is also known that a certain percentage of cancers, particularly breast cancer, are hereditary. These patients may exhibit genetic markers. Mutations of BRCA1 or BRCA2 are found in 80% of these patients and a high percentage of women with mutated BRCA1 or BRCA 2 show a greatly increased risk of developing breast or ovarian cancer. Again, these markers are detected and quantified by blood tests conducted in a laboratory setting. It must be noted that some of these markers are normally present in small amounts in healthy patents and increased levels in cancer patients while others seem to be present only when cancer is present.
U.S. Pat. No. 5,798,266 describes the analysis of mammary fluids for breast disease markers such as described in Porter-Jordan, but particularly addresses carcinoembryonic antigen (CEA), HMFG, MCA, vasopressin or cathepsin D as markers. Also, protein Ig, suggested as a breast cancer marker, has been detected in tears.
Many variations in physiological conditions, genetic defects or disease states result in the release or increase of specific biological and/or chemical moieties, ie., marker compounds, into body fluids, particularly blood and urine. I have now documented several of these markers are present in apocrine sweat and are present in apocrine sweat in elevated levels when compared to blood. These markers can be collected and/or detected and used as an indicator that a disease state or particular physiological condition exists. In a first embodiment, the invention contemplates a skin patch to collect sweat, the patch including monoclonal antibodies or other chemical compounds that can indicate the presence of specific markers. This may include indicators which render visible, the existence of a reaction between the monoclonal antibody or chemical compound and the marker compound to indicate the existence of the condition being tested for. In a second embodiment a wipe to collect sweat may be used instead of a patch. A third embodiment uses an electronic sensor to collect and sample vaporized apocrine sweat or aroma emitted from the apocrine sweat gland. Various different disease states can be detected including, but not limited to heart disease, cancer, autoimmune disease, infectious disease, renal disease, drug intoxication and other systemic illnesses. In particular, occult cancers of the breast, colon, stomach, pancreas, lung, uterus, prostate, bile duct and ovary and neueroblastoma as well as various forms of leukemia can be detected by this technique.
It is generally believed that human sweat has a chemical composition which reflects chemical changes in blood. In other words, when a chemical compound not normally present in blood, or a compound with cyclical presence, appears in the blood stream of an individual, it is believed that that compound, or a corresponding compound, analog, metabolite or reaction product, referred to as a by-product, of that compound which can be correlated with a systemic disease state or a change in physiological function, such as ovulation, can usually be identified in sweat of that individual. However, it has now been discovered that this is not generally so.
The human body has two different kind of sweat glands which, while having some similar functions, also now appear to have certain functions which are completely different. The eccrine sweat glands which are present over much of the body (arms, legs, feet, hands, abdomen, chest, back, forehead, etc.) excrete large quantities sweat and have a primary function of maintaining proper body temperature as a result of the cooling effect which accompanies evaporation of perspiration. On the other hand the apocrine sweat glands, which are located under the arms (the axilla) and in the pubic and mammary areas have a unique and unexpected function and morpohology which is not demonstrated by the eccrine sweat glands. The morphology of the apocrine gland is very important. Every apocrine gland opens at the base of a hair cell. This means that the contents of the apocrine gland are deposited on a hair shaft and that the hair shaft acts as a saturated wick. The warm enclosed space of the under-arm provides a microenvironment which causes vaporization of the apocrine gland secretions. This vapor persists in the armpit which acts as a persistent smell chamber.
The apocrine gland also behaves differently from the eccrine sweat glands in how it responds to certain body conditions. The response to elevated prostate specific antigen (PSA) levels is an example. As set forth in Example 1 below, when the serum PSA level is normal, for example less than 1.0 ng/ml, the apocrine PSA level is essentially insignificant. However, when the serum prostate specific antigen level is abnormally elevated the PSA in apocrine sweat increases to a significantly greater extent and to a significant therapeutically abnormal range. An active transport ATP dependent mechanism takes place to concentrate PSA in the apocrine secretion. As a result of the morphology of the apocrine gland, the apocrine gland functions to saturate the hair follicle cells with PSA transmitting a message detectable by smell as a result of the PSA levels being elevated to an unhealthy level in the armpit smell chamber. With the discovery of this phenomena, readily readable tests providing an early warning of this serious medical problem can be developed.
I have now found that apocrine sweat glands contain chemicals in concentrated quantities which are also uniquely related to disease or physiological state. Such function or capacity is not demonstrated by the eccrine sweat glands. In other words, apocrine sweat emit odors or aromas and contain chemicals which can be directly correlated with disease state or the state of certain body functions which have, in the past only been determined by blood or urine tests or other more complicated diagnostic procedures. In comparison, eccrine sweat does not contain these chemical compounds or, if present, they exist in much lower concentrations. It is believed that this function is part of the normal evolutionary development of humans and provides to a second individual the capacity to sense if the first individual has a disease or body malfunction. The utilization of the function of the apocrine sweat gland as an olfactory signaling system to communicate the health or physiological status of an individual has profound implications for the understanding of evolution in humans, and presumably other animals as well. It is unlikely that the only evolutionary pressure which shaped this system is mate selection. Once the range of physiologic conditions that the apocrine gland is sensitive to is identified it may become clearer. However, the demonstration of an olfactory system to signal health status means there is an evolutionary advantage in identifying who is ill. Caring for the sick is a human trait that confers significant evolutionary advantage. It is entirely possible that humans in pre-historic times, who did not wash and hide their smells behind deodorants may have actually been better than modern man in diagnosing each other""s illness.
These by-products can also be detected by instrumental means or other indicators early in the course of the disease to allow diagnosis while the disease is readily treatable. Apocrine sweat has been discovered to be the body fluid that has evolved for the purpose of communicating the health status of an individual. Also, female apocrine sweat aromas emitted during ovulation may provide a stimulation toward intercourse and procreation; male apocrine sweat aroma is sensed by a female in the selection of a suitable mate for breeding, specifically in terms of the immune system of the offspring.
Several biomarkers have been identified for breast cancer. These can be utilized in an apocrine sweat patch test to signal occult breast cancer. It is believed that anaysis of apocrine sweat will lead to the discovery of entirely new and as yet unknown biomarkers for various cancers. A patch can also be produced which can indicate the presence of more than one marker for more than one disease. Once a biomarker is identified it can be used in immunohistopathological evaluation of malignant tumors to determine its location in the tumor cell.
Once a biomarker is identified an apocrine sweat diagnostic modality, using the teachings herein, can be established. This will result in a diagnostic screening test using the patch and then a secondary confirmatory test can be performed to locate the tumor in the body.
The availability of an apocrine test patch incorporating markers identified as indicative of breast cancer can supplement or be used as a prescreen for mammogram. It is expected that there will be a number of individuals considered by mammography to be normal who will actually have occult breast cancer. These instances, when detected by an apocrine skin patch, should not be considered as false positives for breast cancer but instead an early warning of the future appearance of the disease. Mammography is limited in its ability to detect very small or dispersed tumors. However, tumors of any size generate biological markers, which will be present in the blood and concentrated in the apocrine sweat. The presence of the marker is indicative of an abnormal physiological state whether or not such condition can be determined by other presently available less sensitive diagnostic modalities. Breast cancer now occurs in one out of nine women and is generally considered to have been present for five or six years before presentation (detection by present detection modalities). It is generally accepted that a mammogram has limited sensitivity and that there are individuals who have occult breast cancer that the mammogram will not detect.